PURIFICATION AND BINDING ANALYSIS OF THE NITROGEN-FIXATION REGULATORYNIFA PROTEIN FROM AZOSPIRILLUM-BRASILENSE

NifA protein activates transcription of nitrogen fixation operons by t he alternative sigma(54) holoenzyme form of RNA polymerase. This prote in binds to a well-defined upstream activator sequence (UAS;) located at the -200/-100 position of nif promoters with the consensus motif TG T-N-10-ACA. NifA of Azospirillum brasilense was purified in the form o f a glutathione-S-transferase (GST)-NifA fusion protein and proteolyti c release of GST yielded inactive and partially soluble NifA. However, the purified NifA. was able to induce the production of specific anti -ii. brasilense NifA-antiserum that recognized NifA from A. brasilense but not from K, pneumoniae. Both GST-NifA and NifA expressed from the E. coli tac promoter are able to activate transcription from the nifH DK promoter but only in an A. brasilense background. In order to inves tigate the mechanism that regulates NifA binding capacity we have used E. coli total protein extracts expressing A. brasilense nifA in mobil ity shift assays. DNA fragments carrying the two overlapping, wild-typ e or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size. These data show that the binding activ ity present in the C-terminal domain of A. brasilense NifA protein is still functional even in the presence of oxygen.